We are designing the methods for absolute quantification, developing and testing standards of tagged proteins (in collaboration with Dr. Iain Fraser) and designing peptides for AQUA method of MS-based quantification. The experimental model is the osteoclast development from macrophages. Understanding the mechanisms of osteoclast formation and action is crucial for the progress in the studies of rheumatoid arthritis and osteoporosis. We use well characterized murine macrophage RAW 264.7 cell line. The cells fuse to form multinucleated osteoclasts when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL), but the differentiation process is inhibited by sphingosine-1 -phosphate (S1P). There are changes in protein expression connected with macrophage differentiation into osteoclasts. The mRNA levels of many proteins change and we want to see if these changes are reflected in the changes of the cell proteome. We have optimized the cell culture conditions and osteoclast enrichment. Using SILAC (stable isotope labeling with amino acids in cell culture) we compared the proteomes of untreated RAW 264.7 macrophages, intermediate osteoclasts and differentiated, multinucleated osteoclasts. We are doing the same for macrophages treated with RANKL/S1P combination. The analysis revealed a set of differentially expressed proteins, which we used to design a set of standard peptides for absolute quantification by mass spectrometry. We have performed also mRNA expression analysis using microarrays and identified major differences between all three cell types. Specifically, we found evidence that compared to osteoclast precursors, multinucleated osteoclasts conserve energy by down-regulating pathways involved in cell cycle control, gene expression, and protein synthesis. Similarly to previous reports, multinucleated osteoclasts were found to express relatively high levels of V-ATPase, TRAP, cathepsin K, and integrins. Proteins involved in ATP synthesis and catabolism, localized primarily in the mitochondria, were also up-regulated in multinucleated osteoclasts, suggesting that osteoclasts up-regulate ATP production compared with osteoclast precursors and intermediate osteoclasts. We have confirmed that both mitochondrial mass and potential are elevated in mature osteoclasts, and median mitochondrial protein expression was significantly higher than the median protein expression in other organelles. A set of mouse chemotaxis pathway target proteins was constructed from a literature and pathway database search. Selection of the target peptides used a wide variety of criteria including peptide proteotypic qualities, sequence uniqueness, and vulnerability to modification (e.g., oxidation and deamidation), eliminating many theoretically possible peptides, which could be non-compatible with the mass spectrometric analysis. We used the quantitative data obtained from osteoclast precursors by shotgun proteomics to find the peptides amenable to the analysis in our Orbitrap Velos. SPOT synthesis was used to prepare a set of 409 standard, synthetic peptides which we used to assess the protein expressions in macrophages and osteoclasts. SRM of RAW264.7 cell lysates spiked with the standard peptides resulted in the confident identification and semi-quantitation of 208 of the 409 peptide targets from proteins in the chemokine signaling network. The Multiple Reaction Monitoring (MRM) analysis of a narrower set of 65 heavy-labeled, quantitated internal peptide standards from proteins differentially expressed under different experimental conditions will provide absolute numbers of molecules. Additionally, a supplementary set of 145 crude, unlabeled peptides was obtained to target the missed proteins. Quantitative pathway simulation will be performed using Simmune Modeler (in collaboration with Dr. Martin Meier-Schellersheim). We are using the MRM approach in the collaboration with Dr. Rajat Varma on the exploration of the commonality of gamma chain in interleukin receptors. We have designed and received a set of 77 T-cell signaling-specific peptides and are testing them for use in this project.